Eustoma is a generic name for autogamous seed-propagated plants of the genus Eustoma in the family Gentianaceae and grows natively in regions of southern North America to northern Central America. These plants include two species: (1) Eustoma grandiflorum (English name: Prairie gentian, former scientific name: E. russellianum (Hook) G. Don ex Sweet or Lisianth(i)us russellianus Hook.) and (2) Eustoma exaltatum (English name: Seaside gentian or Catchfly gentian, former scientific name: E. selenifolium Salisb.). The plants are also known familiarly as Lisianthus. In 1835, the native species was brought to Scotland and designated as Lisianthus russellianus Hook. The plants were introduced to Japan in the 1930s and have been actively bred since then, mainly for cut flowers or potted plants. Eustoma grandiflorum is mainly used in such breeding.
Eustoma is known for flowers and ornamental plants of high ornamental and market value. Particularly, varieties having diverse characters have been produced as varieties for cut flowers, and these Eustoma varieties are regarded as one of the principal cut flower species (see Non Patent Literature 1).
In general, first filial generation (F1) plants superior in character to their parents are utilized in major horticultural crops because of the benefits from heterosis. Among Eustoma cultivars highly demanded as cut flowers, first filial generation varieties are also the mainstream because this allows the plants to have disease resistance or high quality.
For efficient seeding in plant production, breeders are required to supply highly pure seeds. The seed production of a first filial generation variety first involves manual emasculation of a seed parent followed by pollinating the stigma of the emasculated seed parent with the pollen of a pollen parent.
The period when a flower of the seed parent can be successfully emasculated is usually limited to a stage immediately before flowering, and also tends to depend on weather conditions. The emasculation therefore presents operation problems in that the possible period of emasculation is rarely predictable. Since this operation is manually performed, pollen that has remained due to insufficient emasculation may cause the emergence of unintended self-pollinated seeds. Contamination by such self-pollinated seeds results in unfavorable reduction in the quality of seeds. For these reasons, there has been a demand for the development of a method for seed production of Eustoma by use of male sterility that eliminates the need of the manual emasculation of seed parents.
Dwarf Lisianthus transformed by rolC gene transfer using Agrobacterium rhizogenes is known (Non Patent Literature 2), which reportedly has small flowers and loses pollen fertility at the same time with the disappearance of apical dominance (rol syndrome). The technique of Non Patent Literature 2 has been applied to a disclosed method for producing an F1 variety using Lisianthus having male sterility, wherein the variety is maintained by adventitious shoot regeneration (Patent Literature 1). The F1 variety produced according to the method, however, possesses undesired characters. Therefore, the method cannot be used in the breeding of cut flower varieties required to have marketable plant postures or heights.
Cytoplasmic male sterility (CMS), one type of male sterility, is cytoplasmically inherited. The crossing of a cytoplasmic male sterile line with a male fertile line supplies highly pure seeds. In addition, the cytoplasmic male sterile line can be crossed with a maintainer line having the same nuclear genome as that of the cytoplasmic male sterile line and a normal cytoplasm to thereby easily maintain and propagate the line. Thus, such a method for seed production of a first filial generation variety by use of cytoplasmic male sterility is very highly practical and is used in a large number of major horticultural crops. Nonetheless, a method for producing a cytoplasmic male sterile Eustoma has not yet been reported, though the development of the method for seed production of Eustoma by use of male sterility has been demanded. Accordingly, seed production that requires emasculation operation is still performed in the seed production of Eustoma F1 varieties due to the absence of a seed production system using practical male sterile lines.
Meanwhile, good flower shelf life that achieves the prolonged ornamental period of flowers resulting from delayed petal aging has been demanded. Examples of causes of petal aging include pollination and ethylene formation caused by damage on the stigma or style of a pistil (Non Patent Literature 3).
Eustoma having a deformed pistil, the stigma of which is kept closed to thereby prevent pollination and delay flower aging is known as a Eustoma variety having good flower shelf life (Patent Literature 2).
Improvement in flower shelf life has required breeding a line having short filaments of stamens, or using a plant line having an organ structure changed to prevent pollination on its stigma physically, as in the deformed pistil mentioned above.
As one condition, the prevention of pollen dispersion has been demanded for flowers and ornamental plants of high market value. All current Eustoma varieties, however, have stamens that produce pollen and therefore cause, for example, pollen dispersion, which is unfavorable due to the fouling of petals or clothing.
Although problems associated with flower shelf life, etc. can be solved by use of male sterility, without changing useful characters, such a practical Eustoma having male sterility has not yet been developed.